The broad long-term objective of this proposal is to elucidate the function of tropomyosin in the regulation of striated and smooth muscle contraction. This requires a detailed knowledge of its interactions with actin and troponin as modulated by myosin, Ca2+, and ATP. Fluorescence measurements of probes attached to cysteine groups of tropomyosin, will be used to monitor the cooperative change in state of tropomyosin associated with myosin subfragment 1 binding to the thin filament. Specifically, the measurements will be used to: show that the tropomyosin state is correlated with acto-myosin subfragment 1 ATPase activity, clarify the role of the two heads of myosin, determine the influence of the cardiac and skeletal troponin components on the tropomyosin state, verify that tropomyosin states, and determine the differences in behavior of smooth and striated muscle tropomyosins. Circular dichroism, fluorescence and chemical modification methods will be used to study the mechanism of assembly of the alpha and beta tropomyosin subunits (whose amino acid sequences slightly differ) into dimers (alphaalpha, alphabeta and betabeta). The conformational and regulatory properties of each dimer will be characterized by unfolding/assembly studies using tropomyosin from rabbit and frog striated muscle, chicken gizzard and bovine aorta smooth muscle, and mutated striated and smooth alphaalpha tropomyosin prepared by recombinant DNA techniques. The relationships between subunit amino acid will allow the characterization of normal genetic variation as well as alterations associated with genetic defects. Myosin rod (smooth and striated) unfolding/assembly studies will be continued to clarify conformational changes in specific regions of the rod associated with filament formation and activity. These tropomyosin and myosin conformational studies also provide basic information about folding intermediates and assembly mechanisms applicable to other proteins with similar coiled-coil structures.